1. Field of the Invention
The present invention relates to DNA fragments that confer resistance to protoporphyrinogen oxidase (PPO; EC 1.3.3.4)—inhibiting herbicides onto plants, plasmids and microorganisms that contain these DNA fragments. The present invention also relates to methods of conferring resistance onto plants and plant cells by using genetically engineered DNA fragments that encode PPO. Other aspects of the present invention are plants and plant cells onto which have been conferred resistance to PPO-inhibiting herbicides. Another aspect of the present invention relates to a method for evaluating the inhibitory effects of compounds on PPO activity utilizing microbial systems differing only by the presence of genes encoding PPO resistant or sensitive to said compounds.
2. Description of Related Art
A group of widely-known compounds used as active ingredients of some varieties of commercially- and otherwise-available herbicides exhibit herbicidal activity in the presence of light, but exhibit no herbicidal activity in darkness. This has led to their common designation as light-dependent herbicides. It has recently been shown that these herbicides induce high levels of porphyrin accumulation in plants and algae, and thus they are now designated as “porphyrin-accumulating type herbicides” [Zoku, Iyakuhin-no-Kaihatsu, (translation: “The Development of Medical Drug Products; continuation”) vol. 18; Development of Agricultural Chemicals II, chapter 16, section 16-1, 1993, Iwamura et al., eds., Hirokawa Shoten, Tokyo ) or simply “porphyric herbicides”. It was reported by Matringe et al., (Biochem J. 260:231 (1989) and (FEBS Lett. 245: 35 (1989)) that porphyrin-accumulating type herbicides inhibit isolated protoporphyrinogen oxidase. Thus porphyria herbicides are also called PPO-inhibiting herbicides. Protoporphyrinogen oxidase is commonly found in microorganisms such as bacteria and yeast, plants including algae and animals. This enzyme catalyzes the last oxidation step which is common in both the heme and the chlorophyll biosynthesis pathways, namely the oxidation of protoporphyrinogen IX to protoporphyrin IX (Matringe et al., Biochem J. 260: 231 (1989)).
Bacterial PPOs are thought to be localized in the cytoplasm and the genes encoding bacterial PPOs have been isolated from Escherichia coli (Gen Bank accession X68660:ECHEMGA; Sasarman et al., Can. J. Microbiol. 39: 1155 (1993)) and Bacillus subtilis (Gen Bank accession M97208:BACHEMEHY, Daily et al., J. Biol. Chem. 269: 813 (1994)). Mouse (Gen Bank accession U25114:MMU25114), human (Gen Bank accession D38537:HUMPOX and U26446: HSU26446) and yeast (Ward & Volrath, WO 95/34659, 1996) genes encoding mitochondrial PPO have been isolated. Genes encoding chloroplast PPO have also been isolated from Arabidopsis thaliana and maize (Ward & Volrath, WO 95/34659, 1996).
Like higher plants, the unicellular green alga Chlamydomonas reinhardtii is highly sensitive to PPO-inhibiting herbicides. However, a mutant strain designated RS-3 (Kataoka et al., J. Pesticide Sci. 15: 449 (1990)) shows resistance specifically to PPO inhibitors. This resistance results from a single dominant nuclear mutation (Sato et al., Porphyric Pesticides: Chemistry, Toxicology and Pharmaceutical Applications, Duke & Rebeiz eds., ACS symposium series 559, pp. 91-104, c. 1994 by the American Chemical Society, Washington D.C.). Furthermore, PPO activity in isolated chloroplast fragments from the RS-3 mutant is significantly less sensitive to PPO inhibitors than similar chloroplast fragments from wild type C. reinhardtii (Shibata et al., Research in Photosynthesis Murata ed., Vol. III, pp. 567-570, c. 1993 by Kluwer 5 Academic Publishers, Dordrecht, Netherlands).
Since most crop plants do not exhibit resistance to PPO-inhibiting herbicides, these compounds cannot be used on farmland when such crops are under cultivation. If it were possible to develop crop plants resistant to PPO-inhibiting herbicides, such herbicides could be used for weed control during the growing season. This would make crop management easier, and increase the value of these herbicides in agricultural applications. For this reason, it is desirable to develop a method for conferring resistance to PPO-inhibiting herbicides or porphyrin-accumulating herbicides upon crop plants.